Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases

Abstract

CDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP1a and ATP 2a, have been identified by searching the Arabidopsisdatabase of expressed sequence tags (dbEST). They represent a novelbranch of hitherto uncharacterized plant peroxidases which is only 35%identical in amino acid sequence to the well characterized group ofbasic plant peroxidases represented by the horseradish (Armoraciarusticana/) isoperoxidases HRP C, HRP E5 and thesimilarArabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea. However ATP 1ais 87% identical in amino acid sequence to a peroxidase encoded by anmRNA isolated from cotton (Gossypium hirsutum). As cotton and Arabidopsis belong to rather diverse families (Malvaceae andCrucifereae, respectively), in contrast with Arabidopsis andhorseradish (both Crucifereae), the high degree of sequence identityindicates that this novel type of peroxidase, albeit of unknownfunction, is likely to be widespread in plant species. The atp 1 and atp2 types of cDNA sequences were the most redundant among the 28 differentisoperoxidases identified among about 200 peroxidase encoding ESTs.Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showedthree identical nucleotide substitutions. This variant form isdesignated ATP 1b. Similarly, six out of totally 16 EST sequences codingfor ATP 2 showed a number of deletions and nucleotide changes. Thisvariant form is desigated ATP 2b.The selected EST clones are full-length and contain coding regions of993 nucleotides for atp 1a, and 984 nucleotides for atp 2a. Theseregions show 61% DNA sequence identity. The predicted mature proteinsATP 1a, and ATP 2a are 57% identical in sequence and contain thestructurally and functionally important residues, characteristic of theplant peroxidase superfamily. However, they do show two differences ofimportance to peroxidase catalysis: (1) the asparagine residue linkedwith the active site distal histidine via hydrogen bonding is absent;(2) an N-glycosylation site is located right at the entrance to the hemechannel.The reverse transcriptase polymerase chain reaction (RT-PCR) was used toidentify mRNAs coding for ATP 1a/b and ATP 2a/b in germinating seeds,seedlings, roots, leaves, stems, flowers and cell suspension cultureusing elongation factor 1α (EF-1α) for the first time as apositive control. Both mRNAs were transcribed at levels comparable toEF-1α in all plant tissues investigated which were more thantwo days old, and in cell suspension culture. In addition, the mRNAcoding for ATP 1a/b was found in two day old germinating seeds. Theabundant transcription of ATP 1a/b and ATP 2a/b is in line with theirmany entries in dbEST, and indicates essential roles for these novelperoxidases.